With the advent of the possibility to generate protein products by recombinant DNA methods and specifically the production of insulin A-chain and insulin B-chain by such techniques [see Goeddel et al., Proc. Nat'l. Acad. Sci. USA 76, 106-110 (1979)], the need for an efficient method for combining the A- and B-chains to form insulin has greatly increased.
Typically, the prior art methods for producing insulin by combination of A- and B-chains use as starting materials such A- and B-chains in the form of their stable S-sulfonates. In general, the A- and B-chain S-sulfonates, either separately or together, are reduced to their corresponding --SH compounds, customarily using a large excess of thiol reducing agent. The products are isolated from the reducing medium and, if not reduced together, are brought together in an oxidizing medium, e.g., air, to achieve combination of A- and B-chains with production of insulin. Examples of this methodology are found in Du et al., Scientia Sinica 10, 84-104 (1961); Wilson et al., Biochim. Biophys. Acta 62, 483-489 (1962); Du et al., Scientia Sinica 14, 229-236 (1965); Kung et al., Scientia Sinica 15, 544-561 (1966); Kexue Tongbao (Republic of China) 17, 241-277 (1966); and Markussen, J. Acta Paediatrica Scandinavica, Suppl. 270, 121-126 (1977).
A modification of this approach involves reduction of the A-chain S-sulfonate followed by reaction of the reduced A-chain with B-chain S-sulfonate in an oxidizing atmosphere. See, e.g., Katsoyannis et al., Proc. Nat. Acad. Sci. (U.S.A.) 55, 1554-1561 (1966); Katsoyannis, Science 154, 1509-1514, (1966); Katsoyannis et al., Biochemistry 6, 2642-2655 (1967); U.S. Pat. No. 3,420,810; and Jentsch, Journal of Chromatography 76, 167-174 (1973).
Another modification involves partial oxidation of the A-chain --SH compound to produce disulfide formation between the A-6 and A-11 cysteine residues followed by oxidation of the product with B-chain --SH or B-chain S-sulfonate. See, e.g., Belgian Pat. No. 676,069 and Zahn et al., Liebigs Ann. Chem. 691, 225-231, (1966).
In each of the above prior art methods, one element is common i.e., the production of insulin by two independent, sequential steps, namely, reduction of S-sulfonate to --SH followed by oxidation to --S--S--.
Dixon et al., Nature 188, 721-724 (1960) describe conditions which suggest single solution conversion of A- and B-chain S-sulfonates to insulin using a thiol reducing agent and air oxidation. The details are quite sketchy, and the yield, based only on activity of product recovered, represented 1-2%. However, Dixon, in Proc. Intern. Congr. Endecrinol. 2nd London 1964, 1207-1215 (1965), in somewhat further elaboration, suggests, in Table IV at page 1211, that the conditions reported in the earlier publication involve separate reduction and oxidation steps.
In distinction to the above prior art methods, it has now been discovered that it is possible under defined reaction conditions to achieve attractive levels of production of insulins or analogs of insulins from S-sulfonated A- and B-chains by conducting both the reduction and oxidation reactions in a single-step, single-solution process. It is to such a process that this invention is directed.